The United States government has rights in this invention by virtue of National Institute of Health grant No GM15539.
In bacteria, operons for amino acid biosynthetic enzymes are controlled specifically by their respective end-product amino acids. In yeast, however, many of the genes encoding amino acid biosynthetic enzymes are subject to regulation by the general amino acid control system. This regulatory network consists of a hierarchy of proteins whose function is to modulate the levels of a number of amino acids in the cell. Starvation for any one of these amino acids leads to derepression of all of the genes subject to general control.
Previous studies of enzymes involved in the biosynthesis of leucine in S. cerevisiae revealed a combination of both general and specific amino acid regulation. LEU4, which encodes the first enzyme in the pathway, alpha-isopropyl malate synthase, abbreviated alpha-IPM synthase, is subject to regulation by both the general amino acid control system and specifically by leucine through feedback inhibition. LEU1 and LEU2, which encode the second and third enzymes in the pathway, respectively, are only under specific amino acid control. Expression of both genes is repressed by elevated levels of leucine. This sensitivity to a specific amino acid seems to distinguish these genes from most others that are associated with amino acid biosynthesis in yeast.
The leucine-specific control of LEU1 and LEU2 is thought to be indirect. Expression of these two genes appear to be a function of the level of alpha-IPM, the product of the first enzyme in the pathway. This is based on the following results: (1) the levels of the LEU1 and LEU2 gene products are sharply decreased in a strain that lacks a functional synthase; (2) leu1 and leu2 mutants, which are expected to accumulate intermediates in the pathway (such as alpha-IPM), exhibit increased levels of beta-IPM dehydrogenase and alpha-IPM isomerase, the products of the LEU2 and LEU1 genes, respectively; and (3) a strain that contains a feedback resistant alpha-IPM synthase and produces high levels of alpha-IPM, also has increased levels of the LEU1 and LEU2 gene products, as reported by Baichwal et. al., in Current Genetics 7, 369-377 (1983) and Brisco et al in Genetics 115, 91-99 (1987). Because alpha-IPM synthase is feedback inhibited by leucine, the levels of alpha-IPM are directly related to the levels of leucine in the cell. Accordingly, if alpha-IPM functions as an inducer, it could be responsible for the indirect, leucine-specific control of LEU1 and LEU2.
A single genetic locus having the potential for being a factor in the regulation of LEU1 and LEU2 was uncovered by analysis of mutants defective in leucine biosynthesis. This mutant allele, designated leu3, is a leaky leucine auxotroph which produces low levels of both the LEU1 and LEU2 gene products and is described by Kohlhaw in "Regulation of Leucine Biosynthesis in lower Eukaryotes", Amino Acid Biosynthesis and Genetic Regulation, L. H. Herrmann and R. L. Somerville, eds., 285-299 (Addison-Wesley 1983). One hypothesis is that the LEU3 gene product functions as a positive activator of LEU1 and LEU2, in conjunction with alpha-IPM as an inducer.
The nucleotide sequences of LEU1 and LEU2 have been established and it has been shown that levels of the respective mRNAs are sensitive to intracellular leucine concentrations (Andreadis et al., Cell 31, 319-325 (1982); Hsu and Schimmel, J. Biol. Chem. 259, 3714-3719 (1984); Andreadis et al., J. Biol. Chem 259, 8059-8062 (1984)). This work also established a section of partial nucleotide sequence homology between the 5'-regions of the two genes.
To date, the sequence of LEU3 has not been known, nor its exact role in regulation of amino acid synthesis, specifically, the interaction of the gene product of LEU3 with LEU1 and LEU2.
It is therefore an object of the present invention to provide a further characterization of the role of LEU3 in the leucine-specific regulation of the LEU1 and LEU2 genes.
It is a further object of the present invention to provide the sequence of the LEU3 gene and to define the roles of portions of the sequence involved in DNA binding and gene regulation.
It is a still further object of the present invention to provide a means for inducing or regulating protein synthesis using the product of the LEU3 gene.